CN1062603C - Film reaction technology for producing D-p-hydroxy-phenyl glycine by enzyme method - Google Patents

Abstract

The present invention relates to a film reaction technology for producing D-p-hydroxy-phenyl glycine by an enzyme method. With the film reaction technology, a hollow fibre reactor or flat-plate micro-filtration film reactor or flat-plate ultrafiltration film reactor is used for immobilizing smicroorganism cells containing carbamyl hydrolase or simultaneously containing dihydropyrimidinase; DL-para hydroxybenzene hydantoin is converted into D-p-hydroxy-phenyl glycine. The whole cell is captured and immobilized at one side of the film reactor through the treatment of crosslinking and flocculation, and thus, the half life period of endoenzyme--carbamyl hydrolase can be raised by three to five times. The application of the film reactor realizes the separation of reaction liquid and microbe cells, and the microorganism cells can be used repeatedly several times to reduce pollution caused by discarding microorganism cells.

Description

The film reaction method of Production by Enzymes D-D-pHPG

The present invention is a kind of film reaction method of being used for fixing cells produce D-D-pHPG.

D-D-pHPG (D-p-HPG) is a main side chain of producing semisynthetic penicillin and semi-synthetic cynnematin, and its synthetic method has chemistry to split and enzyme process splits two classes.Because problems such as chemical method exists cost higher, and is seriously polluted, thereby the enzyme process fractionation comes into one's own day by day.The ultimate principle that enzyme process splits is to utilize the directed D-para hydroxybenzene glycolylurea (D-p-HPH) that transforms of the microorganism cells that contains dihydropyrimidinase to be D-carboxamide D-pHPG (D-N-Carbamyl-p-HPG); Utilize the microorganism cells that contains carbamyl hydrolysis enzyme that it is hydrolyzed to D-D-pHPG (D-p-HPG) again.Under reaction conditions, the spontaneous racemization of L-para hydroxybenzene glycolylurea is a D-para hydroxybenzene glycolylurea, reaches the conversion fully of DL-para hydroxybenzene glycolylurea.Two kinds of enzymes can be contained in respectively in two kinds of microorganism cellss, and also can adopt the microorganism cells that contains two enzymes simultaneously to make two step enzyme reactions once transform DL-para hydroxybenzene glycolylurea is the D-D-pHPG.At present, the subject matter that enzyme process splits is that the vigor of the contained carbamyl hydrolysis enzyme of employed microorganism cells is on the low side, and the transformation period is too short.Cause the intermediate product hydrolysis time long, transformation efficiency is on the low side.Because it is on the low side that cell contains enzyme activity, adopt conventional immobilized cell technique can increase enzyme stability alive, but being even lower, the work of immobilized cell enzyme is difficult to use.From microorganism cells, separate, extract carbamyl hydrolysis enzyme and make existing use of technology of immobilized enzyme, but the cost of enzyme is too high, and product yield is also on the low side.

The method that the purpose of this invention is to provide a kind of D-of preparation D-pHPG adopts this technology to produce the D-D-pHPG, pollutes less, yield height, production cost be low.

For achieving the above object, the present invention combines realization response and isolation integral with film reaction technology with Production by Enzymes D-D-pHPG technology.Feature specifically of the present invention is to adopt tubular fibre or dull and stereotyped micro-filtration, ultrafiltration membrane reactor, immobilization contains carbamyl hydrolysis enzyme or contains the microorganism cells of dihydropyrimidinase simultaneously, can improve the stability of carbamyl hydrolysis enzyme, transforming DL-para hydroxybenzene glycolylurea is the D-D-pHPG.This process for fixation can not lose the vigor of carbamyl hydrolysis enzyme, and can live according to enzyme in the microorganism cells and just regulate the load density of immobilized cell at the membrane reactor inner cell, so that enough enzyme activities to be provided, catalytic hydrolysis D-N-Carbamyl-p-HPG is D-p-HPG.

Adopt the technology of membrane reactor immobilized cell of the present invention, in reaction process, adopt the press filtration operator scheme, but periodical operation, but also operate continuously.The reaction conditions that is adopted will according to the catalytic kinetics behavior of microorganism cells routinely technology design, regulate and control.For the microorganism cells that contains two enzymes, also the regulating and controlling of the reaction conditionss such as permeation flux that technology will be by film routinely is complementary the apparent vigor of dihydro-pyrimidin ferment and the vigor of carbamyl hydrolysis enzyme.Avoid the accumulation of D-N-Carbamyl-p-HPG, realize the high yield conversion of DL-p-HFH to D-p-HPG.

The used membrane reactor of the present invention can be commercial tubular fibre or plate micro-filtration, ultra-filtration membrane separator, also can make up the various forms of membrane reactors that micro-filtration, ultra-filtration membrane that processing made by various materials are formed voluntarily.The aperture of film is a criterion to hold back microorganism cells fully in the reactor.

The biological catalyst that is immobilized in the membrane reactor is a part that contains carbamyl hydrolysis enzyme or contain all kinds of bacterial strains of screening naturally, mutagenic strain, gene recombination bacterial strain or the above-mentioned bacterial strains of dihydropyrimidinase simultaneously, also can be corresponding immobilized enzyme.

Treat to send into membrane reactor one side after immobilized microorganism cells will be handled by linking agent or flocculation agent, under pressure promoted, liquid saw through film and discharges, and cell is fixed in the membrane reactor because of holding back.Used linking agent can be glutaraldehyde, bis-diazotized benzidine-2,2 '-disulfonic acid, 1,5-two fluoro-2,4-dinitrobenzene, adipimide dimethyl phthalate and polyamines, or coupling; Used flocculation agent can be polyacrylamide, poly carboxylic acid, poly-ethylamine, polylysine, calcium chloride, calcium oxide, secondary calcium phosphate, or coupling.During processing the density of cell be 0.8～5% (wet bacterium is heavy/V), crosslinker concentration is 0.01～12.5% (heavy to warm bacterium), and treatment temp is 0～30 ℃, and the concentration of PH4.0～9.0. flocculation agent is 0.1～10% (heavy to wet bacterium), treatment temp is 20～40 ℃, PH5.0～9.0.

The operating process of film reaction of the present invention is to pump in membrane reactor immobilized cell one side by filter screen with the press filtration operator scheme with having not molten particulate DL-para hydroxybenzene glycolylurea solution in the solution storage tank, reaction solution is product through rework solution basin behind the film until substrate conversion.The reactive system nitrogen protection, 30～40 ℃ of temperature of reaction, P6.5～9.0.

The membrane reactor operating method can adopt batch formula operation or operate continuously.Adopt batch formula operation, can after discharging feed liquid, feed intake once more and react.Below by example technology of the present invention is given to illustrate further.

The experiment of example 1. thalline flocculation treatment

The pseudomonas fermented liquid (containing bacterium weight in wet base 3.5%) that contains two enzymes is transferred PH8.0.Get polyacrylamide, 45mgCaCl that two parts of 30ml add 12mg molecular weight 9,000,000 respectively ₂, other gets the original bacteria liquid that a 30ml does not add any material.Handle the centrifugal collection thalline of 1hr through 33 ℃, send into three hollow fiber membrane reactor exocoels (reactor φ 10 * 100 hollow-fibre membrane molecular weight cut offs are 70,000) respectively after the washing once, then under nitrogen promotes, Continuous Flow is crossed 34 ℃ of 0.05%DL-para hydroxybenzene glycolylurea solution (1/15M PH8.0 phosphoric acid buffer) respectively, measures from the liquid sampling that reactor flows out.Thalline, CaCl after former bacterium, polyacrylamide are handled ₂The transformation period of the thalline after the processing was respectively 1.5 days, 0.8 day, 4.5 days.

Example 2 operate continuously mode membrane reactor transformation experiments

(3.1% wet bacterium weight/V) directly send into (reactor φ 26x220, hollow-fibre membrane molecular weight cut off are 30,000) in the hollow fiber membrane reactor, the reaction system cumulative volume is 500ml to pseudomonas fermented liquid 4L.DL-p-HPH to 3.273% transforms and produces D-p-HPG.Conversion condition PH8.0,35 ℃, nitrogen protection earlier with intermittent mode, transforms in the operate continuously mode then, and periodical operation conversion of substrate 55mmol generates product 45mmol.Operate continuously cotransformation substrate 193mmol generates product 134mmol.The substrate conversion amount of operate continuously, product growing amount are respectively 3.5 times, 3 times of periodical operation.

3 batches of formula operation experiments of example

The pseudomonas fermented liquid is sent in the hollow fiber membrane reactor (φ 35x350, hollow-fibre membrane molecular weight cut off are 30,000) after 0.1% calcium chloride is handled, thalline weight in wet base 82 grams.The DL-p-HPH of 3% (W/V) (purity is 91.4%) is suspended in the phosphoric acid buffer of 1% (V/V) glycerine (PH7.5).

Utilize the press filtration reaction pattern, reaction volume 590ml, 32～34 ℃ of temperature of reaction, PH6.5～7.5, flow velocity 50～100ml/min.7 batches of coreactions, the 80.5g that always feeds intake, every batch reaction time 12～22hr, average response productive rate 96.1%, average product molar yield 75.8%, specific optical rotation are-156 ° ± 2 °.

By above-mentioned example, the present invention and existing process for fixation and single batch operation or fixed-bed operation relatively have following advantage:

1. adopt the membrane reactor fixation of microbial cell, do not lose the original vigor of cell, can make half The phase of declining prolongs 3～5 times, for being repeatedly used of former thalline provides fabulous condition, has reduced The pollution that causes because abandoning useless bacterium.

2. by selecting, regulate and control the operating parameter of reactor, realize that two go on foot reasonable of enzyme activities Join, realize the fully conversion of intermediate product, improve yield and the product quality of product.

3. all membrane reactors are produced D-pHPG, and product and microbial cell are in reaction Obtained in the process separating, omitted the step of centrifugation or plate compression, improved to separate and received Rate.

Claims (2)

1. the film reaction method of a Production by Enzymes D-D-pHPG, it is characterized in that adopting tubular fibre or dull and stereotyped micro-filtration, ultrafiltration membrane reactor, immobilization contains carbamyl hydrolysis enzyme or contains the microorganism cells of dihydropyrimidinase simultaneously, DL-para hydroxybenzene glycolylurea is converted into the D-D-pHPG, makes reaction and isolation integral prepare the D-D-pHPG; Immobilization is to send into membrane reactor with containing the enzyme cell after crosslinked, flocculation treatment, promotes to make liquid filtering by pressure, and cell retention is in membrane reactor; Crosslinked, flocculation treatment is selected glutaraldehyde, bis-diazotized benzidine-2 for use, 2 ＇-disulfonic acid, 1,5-two fluoro-2,4-dinitrobenzene, adipimide dimethyl phthalate or polyamines are linking agent, selecting polyacrylamide, poly carboxylic acid, poly-ethylamine, polylysine, calcium chloride, calcium oxide or secondary calcium phosphate for use is flocculation agent, be made into uniform suspension with microorganism cells, the density of suspension cell be 0.8～5% (wet bacterium is heavy/V), crosslinker concentration is 0.01～12.5% (heavy to wet bacterium), treatment temp is 0～30 ℃, PH4.0～9.0; The concentration of flocculation agent is 0.1～10% (heavy to wet bacterium), and treatment temp is 20～40 ℃, PH5.0～9.0; The operation of reactor is to have not molten particulate DL-para hydroxybenzene glycolylurea solution through behind the filter screen; pump in the membrane reactor with the press filtration operator scheme, reaction solution returns the substrate solution basin, the reactive system nitrogen protection after seeing through film; reaction PH is 6.5～9.0, and temperature is 30～40 ℃.

2. according to the described film reaction method of claim 1, it is characterized in that operating method can adopt batch formula operation or operate continuously.